Project Summary: The goal of this proposal is to perform IND-enabling studies of a significantly safer variant of the anti-cancer biologic drug L-asparaginase. L-asparaginases are enzyme drugs that act to deplete the amino acid asparagine from the blood. Due to toxicity, which is especially pronounced in adults, L- asparaginase treatment is limited to acute lymphoblastic leukemia (ALL), a cancer of white blood cells. One source of toxicity of L-asparaginases is due to their bacterial origin (either from E. coli (Elspar) or Erwinia chrysanthemi (Erwinaze)), making the naked drugs highly immunogenic. The current standard of care is a PEGylated version of Elspar called Oncaspar. While PEGylation reduces, but does not eliminate the immunological challenge of using these drugs, the other toxicity-causing factor remains - this being their L- glutaminase coactivity. Therefore, Oncaspar is limited to ALL, where even its use to treat adult ALL patients is highly limited. Of note, L-asparaginase-associated side effects prevent the use of this unique cancer drug in other hematological malignancies (e.g. acute myeloid leukemia) and in solid tumors (e.g. pancreatic cancer), despite strong evidence that L-asparaginases would be effective in treating those cancers. Hence, there is a clear unmet need for an L-asparaginase with reduced immunogenicity and that lacks L-glutaminase coactivity. Recently, we characterized a guinea pig L-asparaginase (GpA) that possesses the required low KM property for clinical efficacy and that exhibits in vivo tumor cell-killing. Notably, we also discovered that GpA is devoid of the toxicity-causing L-glutaminase co-activity. With ~70% sequence identity to human L- asparaginase, GpA should be less immunogenic compared to the bacterial enzymes that share only ~25% sequence identity with the human enzyme. We recently identified the lead biologic GpA369 which is a stable and active C-terminal truncation of GpA comprising the catalytic domain. Here we will perform the required studies required to bring this novel enzyme drug to patients. In Aim 1 we will increase its sequence identity to the human homolog using a structure-guided approach and identify the optimal PEGylation strategy. Aim 2 will determine the pharmacokinetic properties of the top 3 optimized leads from Aim 1, as well as confirm their anti- cancer efficacy in a human ALL mouse model. In Aim 3, the top variant (optimal combination of PK and anti- cancer efficacy) will proceed to toxicity studies, first in mice, followed by more extensive studies in rats and dogs. Finally, Aim 4 will evaluate the immunogenicity of our enzyme drug in a novel mouse model that has a reconstituted human immune system, and compare our drug to Oncaspar. Together, these studies will bring us to the cusp of submitting an IND application for testing this novel L-asparaginase in patients. Impact is predicted to extend beyond ALL, since the improved safety profile of our L-asparaginase variant would enable its use in multiple cancers for which effective options are sorely lacking and which strong data suggests that an L-asparaginase drug would be effective but is not used due to the unacceptable toxicity profile of current L- asparaginase options.